Abstract
We examined the effects of two novel 1alpha,25-dihydroxyvitamin D3-26,23-lactone (1alpha,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analogues. We found that the 1alpha,25-lactone analogues (23S)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S, 25R)-1alpha,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10(-6) M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10(-7) M) was found to be an effective antagonist of both 1alpha,25(OH)2D3 (10(-8) M) mediated induction of p21(WAF1, CIP1) in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha, 25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR.
Highlights
We examined the effects of two novel 1␣,25-dihydroxyvitamin D3-26,23-lactone (1␣,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1␣,25-dihydroxyvitamin D3 (1␣,25(OH)2D3) and its analogues
We found that the 1␣,25-lactone analogues (23S)-25-dehydro-1␣-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1␣-hydroxyvitamin-D3-26,23lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S,25R)-1␣,25(OH)2D3-26,23lactone, but did not induce cell differentiation even at high concentrations (10؊6 M)
TEI-9647 (10؊7 M) was found to be an effective antagonist of both 1␣,25(OH)2D3 (10؊8 M) mediated induction of p21WAF1,CIP1 in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR
Summary
Chemicals—25-Hydroxyvitamin D3 (25(OH)D3), 1␣,25(OH)2D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 1␣,25(OH)2D3-26,23-lactone, and its analogues (TEI-9616, TEI-9647, and TEI-9648) were synthesized in our laboratory as described previously [20, 30]. Cells were suspended in serumfree medium, and NBT/TPA solution (dissolved in PBS) was added. ␣-NB esterase activity was measured as follows: cell seeding, treatment, and collection were performed according to the method described above. Transactivation measured by luciferase activities was standardized by the galactosidase activities of the same cells determined by a -galactosidase enzyme assay system (Promega). These plasmids, together with the hVDR expression vector, pSG5hVDR Radioactivity in the effluent mixed with 8 ml of toluene-based scintillation fluid was measured by a Beckman model LS6500 liquid scintillation counter On this system standard 1␣,25(OH)2D3, 24-oxo1␣,25(OH)2D3, 24,25,26,27-tetranor-1␣,25(OH)2D3, (23S,25R)1␣,25(OH)2D3-26,23-lactone and 1␣,24R,25(OH)3D3 eluted at 16.3, 19.3, 23.8, 29.0, and 29.3 min, respectively
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